Haemophilia A and B are inherited X-linked coagulopathies characterized by a lack of coagulation factor VIII (FVIII) and factor IX (FIX) respectively. Haemophilia A affects about 1 in 5 000 and Haemophilia B about 1 in 35 000 male births (1,2). Haemophilia leads to impaired thrombin generation, weak and vulnerable clots, and therefore spontaneous bleeding. Compared to severe patients, bleeding episodes are less frequent in moderate/mild patients, and are usually provoked by trauma or invasive procedures. Current treatment includes replacement therapy with recombinant or purified FVIII or FIX (3). However, one of the most serious drawbacks of these treatments is the development of alloantibodies, called inhibitors, inhibiting the activity of the coagulation factor, in a non-negligible number of patients with haemophilia A: 20-25% of severe patients and 7-13% of moderate/mild patients (4). Treatment of these inhibitor patients is limited to FVIII- or IX-bypassing agents, such as recombinant FVIIa (Novoseven®) or plasma-derived activated prothrombin complex (FEIBA®). However, these products have considerably shorter half-lives (4-7 h for FEIBA® and 1.5-2.7 h for Novoseven®), than the respective half-lives of FVIII (˜12 h) and FIX (˜18 h). These short half-lives require the need for frequent infusions, limiting the use of both products for prophylactic purposes and increasing the costs. They are expensive and a substantial number of patients do not respond to these agents.
Therefore, as an alternative therapeutic approaches, Factor X (FX) is a more attractive bypassing molecule, since it displays a 40 h half-life and is part of the coagulation cascade normally activated by both FVIII and FIX. For instance, a FX variant which combines activation of the molecule by thrombin with a long survival mimicking that of the FX zymogen was disclosed in WO 2010070137. This FX variant is activated without FVIII or FIX, in vitro as well as in vivo and could thus be used as a bypassing agent in both hemophilia A and B. However, the FX variants of the prior art are susceptible to inactivation due to the presence of inhibitors such as Antithrombin (AT) and/or the Tissue Pathway Inhibitor (TFPI). Actually, TFPI in complex with FXa (TFPI)-FXa inhibits the initiating complex of the coagulation Tissue Factor (TF)/FVIIa. TF/FVIIa complex initiates coagulation by activating FX. However, activation of FX to FXa by the FVIIIa/FIXa complex counteracts the activity of TFPI, allowing coagulation to proceed. In patients with haemophilia, the propagation phase of thrombin generation is not sustainable due to the lack of intrinsic tenase (FVIIIa/FIXa) complex (11). The limited availability of thrombin via TF-FVIIa pathway is in part due to rapid inhibition of FXa and thrombin by TFPI and AT (12, 13). In particular, plasma AT levels are normally high (2.6 μM), and AT is capable of inhibiting trace amounts of FXa and thrombin (13, 14). Therefore, a new upcoming strategy to treat coagulation disorders like hemophilia has recently emerged and implies neutralization of natural anticoagulants and especially TFPI and AT. This is based on several observations. Thus, low TFPI and AT levels in neonates are deemed to be important in augmenting thrombin generation with lower levels of procoagulant factors (15, 16). Furthermore, low AT levels improved haemostatic function in FVIII-deficient mice with heterozygous AT deficiency (17). Very recently, a biopharmaceutical company (Alnylam) developed an RNAi therapeutic targeting antithrombin (ALN-AT3) for the treatment of haemophilia and other rare bleeding disorders, which is currently being investigated in a multinational Phase 1 trial in haemophilia subjects. However, because of the high concentration of circulating plasma antithrombin (3-5 μM), targeting antithrombin via inhibitory molecules may not be the ideal way to obtain therapeutic attenuation of coagulation in haemophilia patients. For TFPI, different blocking reagents have been evaluated as possible therapeutic agents in different animal models with haemophilia (18, 19, 20, 21). However, there are some drawbacks for anti-TFPI agents. Indeed, TFPI is distributed among different pools: the major part is located in or at the endothelial surface, while the rest is distributed equally between platelets and plasma. Moreover, only 1% of total TFPI circulates as free protein in plasma, with the remainder bound to LDL-particles. Due to this complex biodistribution, it is difficult to monitor the efficacy of TFPI inhibition upon treatment in patient plasma samples. A second issue is that different splicing forms of TFPI are present (TFPIalpha and TFPIbeta), which act in different ways. Anti-TFPI based therapy should therefore be using agents that differentiate between both forms. To illustrate the difficulty to use the TFPI-blocking strategy, a clinical trial ran by Baxter using an aptamer directed against TFPI has been stopped due to an increased number of bleeding events. Biological explanations for this observation is that the blocking agent releases intracellularly stored TFPI, impacts its metabolism, and prolongs its circulatory half-life.